1. Field of the Invention
The present invention relates to L-tyrosine-producing bacterium and a method for producing L-tyrosine by fermentation.
2. Brief Description of the Related Art
L-tyrosine is useful as a raw material or as a synthetic intermediate for pharmaceuticals. Conventional methods for producing L-tyrosine include extracting L-tyrosine from precipitates obtained during degradation of vegetable proteins, such as soybean proteins (JP07-10821 A), and producing L-tyrosine by fermentation using, for example, bacteria of the genus Brevibacterium (JP09-121872 A).
Bacteria of the genus Brevibacterium have an L-tyrosine biosynthetic pathway called the arogenate pathway, which produces arogenate from prephenate by prephenate aminotransferase, and then produces L-tyrosine from arogenate by arogenate dehydrogenase. Conventionally, L-tyrosine is produced by utilizing a bacterium of the genus Brevibacterium, since in this bacterium, the above-mentioned two enzymes are not inhibited when L-tyrosine is final product. However, growth of this bacterium is slow, and therefore productivity of L-tyrosine by this bacterium is low.
On the other hand, in the L-tyrosine biosynthetic pathway of Escherichia coli, prephenate is dehydrated by prephenate dehydrogenase (hereinafter, abbreviated to “PDH”) into 4-hydroxyphenylpyruvic acid, from which L-tyrosine is synthesized by aromatic amino acid aminotransferase. A problem in producing L-tyrosine by fermentation using Escherichia coli is that PDH, a first enzyme in this L-tyrosine-synthetic pathway, is subject to strong feedback inhibition by L-tyrosine, even with a low concentration of L-tyrosine. To efficiently produce L-tyrosine using Escherichia coli, the desensitization of this feedback inhibition by L-tyrosine is necessary first and foremost.
As for L-tyrosine production by Escherichia coli, it has been reported that bacteria resistant to p-fluoro-tyrosine excrete L-tyrosine (J. Bacteriol. 1958 September; 76(3): 328), and it was also reported that bacteria resistant to β-2-thienyl-alanine (J. Bacteriol. 1958 September; 76(3): 326), and bacteria resistant to P-aminophenylalanine (J. Bacteriol. 1969 March; 97(3): 1234) excrete L-tyrosine. However, mutations carried by those bacteria were not specified, or were found to be in a gene other than the PDH gene, such as 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS) gene or the like. Therefore, neither L-tyrosine-producing bacterium carrying PDH which is desensitized to feedback inhibition, nor a mutation resulting in desensitization of feedback inhibition of PDH activity has been reported.
Although a strain of Bacillus bacterium in which feedback inhibition of PDH is desensitized was obtained from D-tyrosine-resistant strains (J. Biol. Chem. 1970 Aug. 10; 245(15): 3763), no mention has been made as to whether the desensitization of this inhibition actually results in enhanced production of L-tyrosine. Furthermore, a specific site of the mutation has not been examined. Furthermore, the procedure to obtain a D-tyrosine-resistant strain was not applicable to Escherichia coli, because Escherichia coli is inherently resistant to D-tyrosine due to the presence of the dadA (D-amino acid dehydrogenase A) gene (J. Bacteriol. 1994 March; 176(5): 1500).
In Escherichia coli, PDH is encoded by the tyrA gene and is identical to Chorismate mutase (CM) (SEQ ID NO: 1). It has been reported that PDH is inhibited by L-tyrosine at a concentration as low as 300 μM (Biochemistry. 1985 Feb. 26; 24(5): 1116). On the other hand, it was reported that the active sites of PDH are within the region of amino acids at positions 94 to 373, and that the region also includes sites involved in inhibition of its activity by L-tyrosine (Eur J Biochem. 2003 February; 270(4): 757). However, it has not been elucidated which amino acid residue is involved in the inhibition.
3-fluoro-tyrosine is known as an inhibitor of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and has been used to isolate a tyrosine repressor (tyrR)-deficient strain. Although 3-fluoro-tyrosine is also known to inhibit PDH activity, the use of 3-fluoro-tyrosine to obtain a PDH mutant has not been reported to date.